The overall objective of the proposed research is to define the evolutionary diversification of the immunoglobulin heavy chain variable region (VH) gene family at the DNA level. The specific studies proposed will be directed at characterizing the VH gene family in a representative reptilian, Caiman crocodylus. Preliminary studies have documented the ability to detect multiple hybridizing components in restriction enzyme-digested DNA isolated from this species using murine VH probes. The proposed studies will isolate homologous (hybridizable) genetic regions from recombinant DNA libraries constructed in Lambda phage and cosmid vectors. Initially DNA representative of germline configurations will be examined. Physical maps will be constructed and the VH regions will be characterized by DNA sequencing; inter- and intraspecies relationships will be explored by the mathematical method of metric analysis. DNA sequence data will be analyzed in terms of potential restriction sites for defining homologous VH regions which will be used as probes to definitively characterize the VH gene complex in this species and possibly extend the analysis to more distant levels of phylogenetic development. Homologous VH (and heterologous) probes also will be employed to select VH genes which may be present in rearranged configurations in recombinant libraries constructed from normal lymphoid DNA. Subsequent studies will involve characterization of the VH gene family in Tupaia belangerii, and insectivore which occupies an early critical position in the evolutionary radiation of the mammals. Taken together these studies will provide both a novel description of the extent and nature of the VH complex at key points in vertebrate evolution and basic insight into phylogenetic diversification patterns of this unique gene family. Furthermore, the possibility of localizing DNA sequences related to the organization and stabilization of this gene complex may provide unique insight into general mechanisms related to the evolutionary dynamics of other multigenic families.